Fracture patients with a history of chronic alcohol abuse have a higher incidence of delayed unions and non-unions. In experimental models of fracture healing alcohol inhibits new bone formation in the fracture site and promotes fibrogenesis, accumulation of immature cartilage, decreased osteoblast number and function, and decreased stiffness and strength of the repair tissue. Thus, alcohol promotes scar tissue formation instead of bone regeneration in the fracture site by interfering with cellular mechanisms in both the direct (intramembranous) and indirect (endochondral) pathways of new bone formation. Inhibition of new bone formation and the resulting deficiency in fracture healing in rats given ethanol by subcutaneous injection in a binge-drinking protocol was restored to normal healing by injection of the dietary antioxidant n-acetylcysteine (NAC) during two weeks after fracture. Hypothesis. We hypothesize that treatment with the antioxidant n-acetylcysteine (NAC) will restore the balance of oxidative stress and antioxidant response in the fracture site and thus restore the normal fracture healing sequence including the initial inflammatory phase, Wnt/beta catenin signaling, and endochondral and intramembranous ossification to prevent failure of fracture healing in chronically ethanol-fed rats. Specific Aims. (1) Determine the effects of alcohol consumption/ abstinence and restriction of diet intake before and after fracture on restoration of normal bone repair outcome in untreated and NAC-treated rats. (2) Determine whether oxidative stress is increased by both ethanol and reduced diet intake and whether post- surgery abstinence from ethanol and/or NAC treatment will restore the oxidant-antioxidant balance, inflammatory response, Wnt/beta catenin signaling, endochondral ossification, and intramembranous ossification observed in normal fracture healing. Research Design. Rats 9 months of age, which corresponds to average human age of 20 years, will be fed the Lieber-DeCarli ethanol diet daily for 16 weeks. Transverse osteotomy will be performed at middiaphysis of one femur of the rat. The femur will be immobilized by internal fixation. This model will be created in groups of rats that have been given the ethanol diet and ethanol-free (control) diet. In Aim 1, the outcome of healing will be evaluated at 16 weeks post surgery by mechanical testing. The age of the rat at this time will be 17 months, which corresponds approximately to human age of 60 years. We will determine if 14 days of NAC treatment prevents the ethanol-induced inhibition of bone healing and if this result is also achieved merely by stopping the ethanol feeding after surgery and allowing the animals to consume the control diet ad libitum. In Aim 2, we will obtain preliminary data to answer the following questions, which will provide the basis for a full-scale project leading to clinical translation: (1) In fracture healing failure associated with chronic alcoholism how does alcohol-induced oxidative stress affect (a) recruitment of inflammatory cells and their cytokine expressions that are critical for fracture healing? (b) Wnt/beta catenin signaling that mediates new bone formation? (2) What are the specific mechanisms of action of NAC that restore Wnt/beta catenin signaling and new bone formation in the context of chronic alcoholism. PUBLIC HEALTH RELEVANCE: Persons with a long history of chronic and excessive alcohol consumption (alcohol abusers) experience complications often leading to failure of fracture healing. Experimental studies show that alcohol causes cellular abnormalities in the early stage of fracture healing leading to this failure. In the proposed project we will evaluate the efficacy of the antioxidant n-acetylcysteine (NAC) to prevent the failure of fracture healing in alcohol abusers. In a recent study it was found that inhibition of new bone formation in closed femoral fractures created in rats following binge alcohol exposure could be prevented by administration of NAC during 2 weeks after fracture.The efficacy of NAC for clinical treatment of a large variety of conditions has been documented in a large number of clinical studies during the past two decades. It is well tolerated, has well-defined mechanisms of action and is acknowledged as a safe antidote for cysteine/glutathione deficiency .